J. Gen. App!. Microbio!.

, 42, 181-187 (1996)

Short Communication

COMPARATIVE SEQUENCE ANALYSIS ON THE

18S rRNA GENE OF ASPER GILL US OR YZAE,
A. SOJAE, A. FLA VUS, A. PARASI TI C US,
A. NIGER, A. A WAMORI AND A. TAMARA

SAYUKI NIKKUNI,* NAOHARU KOSAKA,' CHISE SUZUKI,

AND KATSUMI MORI

National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries,

Tsukuba 305, Japan

The molds Aspergillus oryzae, A. awamori and A. niger are widely used in the
food industry in Japan. On the other hand, A. oryzae, A. flavus and A. parasiticus
are very closely related species of the A. flavus group (12). Many isolates of A.
flavus and A. parasiticus produce the carcinogenic secondary metabolites, aflato-
xins. Therefore, many taxonomical studies on these species have been carried out
(3,10,12). Recently, the applicability of molecular biological techniques for
bacterial classification such as DNA base composition, DNA homology and se-
quence comparison of 16S ribosomal RNA (rRNA) is now well accepted (5,16).
The sequence comparison of small subunit (16S-like) rRNAs (or rRNA genes) can
provide a means for analyzing phylogenetic relationships (18). Chang et al. (1)
have used partial sequence comparisons of 18S rRNA comprising 558 nucleotides
to determine the evolutionary affinities among eleven species of Aspergillus and
associated teleomorphs. Dupont et al. (2) analyzed the 5' end of the 28S-like
rRNA molecule (about 130 nucleotides) and constructed a phylogenetic tree
computed from the number of nucleotide differences. Recently, a direct sequencing
method of rRNA gene (rDNA) using the polymerase chain reaction (PCR) has
been developed (17). This study was undertaken to sequence the 18S rRNA genes
of A. oryzae, A. awamori and their related species using a direct PCR sequencing
method and to get information on phylogenetic relationships among these species.
A. oryzae ATCC 1011 (=Thom No. 113), A. sojae IFO 4386, A. tamarii JCM
2259, A. niger IFO 6341( =ATCC 6275), A. awamori IFO 4033 (=ATCC 38854),
* Address reprint requests to: Sayuki Nikkuni
, National Food Research Institute, Ministry of
Agriculture, Forestry and Fisheries, 2-1-2 Kannondai, Tsukuba 305, Japan.

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1996 18S r DNA Sequences of Aspergillus oryzae and Its Allies 183
184 NIKKUNI et al. VOL. 42

A. flavus NFRI 1212 (= NRRL 11612) and A. parasiticus NFRI 1153 (= NRRL
2999) were grown in 500 ml Erlenmeyer flasks containing 100ml YM broth (Difco,
Detroit, MI, U.S.A.) at 30°C for 30 h on a rotary shaker (150 rpm). Mycelia were
harvested with filter paper (Toyo No. 2), washed with deionized water, immersed
into liquid nitrogen and lyophilized.
Genomic DNA was isolated from 0.5 g of lyophilized mycelia according to the
methods of Raeder and Broda (11). The 185 rRNA gene was selectively amplified
by PCR using the synthesized oligodeoxynucleotide primers, NS 1 (17) and ITS2
(17), which were shown in Fig. 1, with or without the additional sequence of 5'-
TGTAAAACGACGGCCAGT ( - 21 M 13) at the 5' terminal of the primers, Taq
Polymerase (Perkin Elmer, Foster City, CA, U.S.A.), the PCR buffer (Optimizer
kit, Idaho Technology, Idaho Falls, ID, U.S.A.) and Air Thermo-Cycler 1605
(Idaho Technology). Amplified DNA was purified with Centricon-100 column
(Amicon, Beverly, MA, U.S.A.). The nucleotide sequences of the PCR products
were determined in both directions by the dideoxynucleotide chain termination
method (15) using Taq polymerase and the dye primer (-21M13) (Perkin Elmer)
and using a DNA sequencer (model 373A, Perkin Elmer).
The evolutionary distance (Knuc) between sequences was calculated by
Kimura's formula (6). A phylogenetic tree was prepared by the neighbor joining
(NJ) method (14) from the evolutionary distance data.
The sequences of 1761 nucleotides of the entire genes except for about 40
nucleotides at the 5' ends of A. oryzae ATCC 1011 and A. flavus NFRI 1212 were
determined. The determined 185 rRNA gene sequence of A. oryzae ATCC 1011
was aligned manually with the sequence data of Saccharomyces cerevisiae (13) as
shown in Fig. 1. Unidentified bases are marked as "N" and a gap in a sequence is
marked with an asterisk "*". The sequence of A, oryzae ATCC 1011 had 88.1%
similarity to that of S. cerevisiae in the sequenced region (1761 nucleotides). The
sequences of 18S rRNA gene of A. oryzae ATCC 1011 and A. flavus NFRI 1212
were found to be identical in the sequenced region (39-1789 [S. cerevisiae number-
ing (13)]). Chang et al. (1) have revealed that the sequences of 18S rRNA of A.
oryzae and A. flavus in all sequenced regions (558 nucleotides) were identical.
The sequences of 1733 nucleotides of entire genes except for about 40 nucleo-
tides at the 5' ends and about 30 nucleotides at the 3' ends of A. sojae IFO 4386, A.
parasiticus NFRI 1153, A. tamarii JCM 2259, A. niger IFO 6341 and A. awamori
IFO 4033 were also determined. The sequences of A. sojae IFO 4386 and A.
parasiticus NFRI 1153 were identical with that of A. oryzae ATCC 1011 in the
sequenced region (39-1761). The sequences of A. niger IFO 6341 and A. awamori
IFO 4033 were also identical in the sequenced region (39-1761). Eleven base
substitution points, 178, 248, 708, 1035, 1048, 1057, 1150, 1348, 1499, 1677 and
1703 [S. cerevisiae numbering (13) ] were identified between A. oryzae ATCC 1011
and A. niger IFO 6341 in the sequenced region (39-1761) (Table 1). The sequence
of A. tamarii JCM 2259 differed from the A. oryzae ATCC 1011 sequence at only a
single nucleotide (position 1488 [S cerevisiae numbering (13)]), where "A" in A.
1996 18S rDNA Sequences of Aspergillus oryzae and Its Allies 185

Table 1. Locations of base differences in test strains.

Fig. 2. Phylogenetic tree for Aspergillus oryzae ATCC 1011, A, sojae IFO 4386,
A. tamarii JCM2259, A. niger IFO 6341, A. awamori IFO 4033, A. flavus NFRI 1212
and A. parasiticus NFRI 1153.
The tree was calculated by the NJ method based on sequences of 1704 continuous
nucleotides of 18S rRNA gene. Knuc, relative evolutionary distance, was calculated by
Kimura's formula (6). Sequence data of S. cerevisiae were from Rubtsov et al. (13).

tamarii JCM 2259 was replaced with "G" in A. oryzae ATCC 1011. The sequence
of A. tamarii JCM 2259 differed from that of A. niger IFO 6341 at 12 nucleotide
positions in the sequenced region (39-1761).
The phylogenetic tree constructed by the NJ method (14) is shown in Fig. 2.
There are two major groups. One group includes A. oryzae ATCC 1011, A. sojae
IFO 4386, A. flavus NFRI 1212, A. parasiticus NFRI 1153 and A. tamarii JCM
2259; the other is the A. niger IFO 6341 group including A. awamori IFO 4033. All
the Aspergillus species used in this study are distant from S. cerevisiae.
Klich and Mullaney (7) have found that SmaI digests of total DNA can be
used for differentiation of A. flavus from A, oryzae and have presumed that the
differences in the restriction fragment patterns may be attributed to rDNA se-
186 NIKKUNI et al. VOL. 42

quence difference. Although there is no SmaI site (CCCGGG) in the sequenced

18S rRNA gene region (39-1789), there is one SmaI site 102by downstream of the
3' end of 18S rRNA gene (Fig. 1). Therefore, the differences of SmaI digests may
be attributed to 28S rRNA gene or noncoding region between rDNAs.
Our results show that A. oryzae ATCC 1011 cannot be distinguished from A.
sojae IFO 4386, A. flavus NFRI 1212 and A. parasiticus NFRI 1153 and A. awamori
IFO 4033 also cannot be distinguished from A. niger IFO 6341 based on the
sequence of 18S rRNA gene. It means that a highly conserved gene, like the 18S
rRNA gene, says nothing about closely related strains. However, it can be said at
least that A. oryzae, A. sojae, A. flavus and A. parasiticus are phylogenetically very
close. Kurtzman et al. (8) investigated DNA relatedness among A. flavus, A.
oryzae, A. parasiticus and A. sojae and have shown that all four species have high
(69-100%) nuclear DNA homology and similar genomic size. They have proposed
from these results that the four taxa represent a single species (8). On the other
hand, Dupont et al. (2) revealed the differences in the sequence (about 130
nucleotides) of 28S-like rRNA between A. oryzae and A. flavus. Klich and
Mullaney (7), as mentioned above, found differences in the sequences of DNA
between A. f lavus and A. oryzae. Gomi et al. (4) confirmed this method and applied
it to A. parasiticus and A. sojae. Moody and Tyler (9) have reported that
mitochondria) DNA restriction fragment length polymorphisms (RFLP) clearly
distinguished A.flavus, A. parasiticus and Aspergillus nomius. Guidelines for the use
of these molecular biological techniques have not yet been established in the
taxonomy of filamentous fungi. Further investigations are needed.
The nucleotide sequence data reported in this paper will appear in the DDBJ,
EMBL and GenBank nucleotide sequences databases with the following accession
numbers: D63698/A. oryzae ATCC 1011, D63696/A. flavus NFRI 1212, D63700/A.
sojae IFO 4386, D63699/A. parasiticus NFRI 1153, D63701/A. tamarii JCM 2259,
D63697/A. niger IFO 6341 and D63695/A, awamori IFO 4033.

We thank Dr. Naruya Saitou, National Institute of Genetics, Mishima, for supplying the computer

program for the NJ method.

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